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Acta anat. 104: 104-122 (1979)

Evolution of the pineal gland in the adult chicken

1. Boya and J. Calvo

Department of Histology and General Embryology, Faculty of Medicine, University Complutense, Madrid, Spain

Key words. Pineal . Ren . Evolution . Structure . Ultrastructure . Ristochemistry

Abstract. The structural pattern ofthe pinea) gland in the hencorresponds to a more advanced stage of the evo)ution which began in an early period of the animal's Jife. This evoJution cor­responds mainly to the transformation ofthe large follicular cavities into cellular 'rose ttes'. The parafollicular Jayer disappears from the rosette wall which thus remains with onJy one row of cells (A and B pineaJocytes).

The cellular hypertrophy and the great deveJopment of the pinealocyte organelles in the adult pineaJ g)and makes us think of this gland as a functionally active organ. This functionaJ activity must have remained during the entire period of the time studied (1-5 years), due to the ultra­structural uniformity found and due to the fact that we couJd not observe any type of degenerative process in the g)and.

Introduction

Morphological research on the bird pineal gland has been centred basically on the identification of photoreceptor structures. These structures possess a very rudimentary character in these species [Col/in , 1966a, b, 1967, 1968, 1969, 1971; Oksche ond VOl/pel van Harnock, 1966; Bischoff, 1969; Oksche O/ul Kir· schlein, 1969; Oksche el 01.,1972; Col/in el 01.,1976]. In many of these species, however, there are no de­tailed descriptions of the histological structure of the pineal gland.

In a series of previous papers we have described the structure of the pineal gland during its embryonic development [Calvo and Boya, 1977a-d] and its

post-hatching morphological evolution [Boya and Zamorano, 1975; Boya and Calvo, 1977a, b]. We have as well described certa in particular aspects of it, such as the innervation development [Calvo ond Boya, 1977a-d] and the Iysosomal nature of the polymorph­OllS dense bodies in the B pineaJocytes [Calvo ond Boya, 1977a-d]. We believe that the knowledge of the normal structural pattem of the pineal gland is a previous and necessary requirement in order to inter­pret the possible morphological variations provoked by the different experimental si tuations. Thus, pineal morphology may be an important research tool for the knowledge of pineal function.

In this papel', we study the pilleal structure during adult life from a light-microscopicaJ, histochemical and ultrastructural poiot of view.

105 The pineal gland in the adult chicken

Materials and methods

Hen pineals of 1, 2, 3, 4, and 5 years of age were used for our study. The hens were always maintained under natural conditions of light and feeding. The animals wen; sacrifized by decapitation and the pineal glands were fixed immediately by immersion in the corresponding fixative.

The fixatives used for Iight-microscopical studies were 10% formalin and Bouin's fluid. The samples were embedded in paraffin and serially sectioned at 7 ,um of width. The staining techniques used were HE, Van Gieson, PAS and the Gomori technique for re­ticular fibres.

The histochemical studies were donc in pineals fixed in 0.1 M cacodylate-buffered 3 % glularaldehyde, pH 7.4, during 2 h at 4 oc. After washing Ihe samples in cacodylale buffer, the sections were oblained from a freezing microtome. TIle sections were incubaled 30-45 min al 37"C in .\filler and Palade's [1964) modi­ficalion of Ihe Gomori medium for acid phosphalase. Part of Ihe sections were reduced in arrunonium sulphile for study wilh the light microscope. The unreduced sections were post-fixed and embedded in Vestopal W for study with tlle electron microscope.

For Ihe ultrastructural studies, the pineals were fixed in 0.1 M phosphate-buffered 5% glutaraldehyde, pH 7.4. The post-fixation was done with phosphate­buffered 2% osmium tetroxide. After dehydration in acetone, the samples were embedded in Vestopal W. The sections were stained with uranyl acetate and lead citrate and then studied in an EM 201 Philips micro­scope.

Results

Light microscopy The pineal parenchyma of the adult hen

presents a compact aspect under the light microscope, and there are no large variations along the age intervals studied . The folJicular cavities, typical in young animals, are not found here (fig. 1). Also there is not clear limit between the folJicular and parafollicular layers as found in previous ages (fig. l) . Instead of all this, the parenchymal cells show an apparently homogeneous arrangement. Tbis

gives the parenchyma uniform appearance when observed at low magnification. A de­tailed study, however, proves clearly that the pinealocytes group themselves in radial ar­rangements forming cellular rosettes. The en­tire pineal parenchyma is composed of these rosettes. Each rosette contains only one row of nuclei arranged in a circular fashion. Inside the row of the nuclei, perfectly ¡ined, there is a band with no nuclei corresponding to the supranuclear portion of the cytoplasm of the radially arranged pinealocytes. We could not find lumens in the centre of the rosettes. Occasionally, we found formations of larger diameter that frequently do not arrange them­selves in a circular fashion but rather in a more irregular way. Also, their supranuclear cyto­plasms are broader. The centre ofthese forma­tions may present an empty cavity of small diameter and irregular borders. Sometimes, the centre is totally occupied by the apical cytoplasms of the pinealocytes or by a small group of cells.

The modification of Miller and Palade [1964] of the Gomori technique for acid phos­phatase shows the existence of a great amount of Jysosomes in the pineal parenchyma of the adult hen (fig. 2). All the pineallysosomes place themselves in circular arrangements or lyso­somal 'rosettes' whose size and distribution are parallel to the cellular rosettes observed with aniline techniques.

The stroma of the adult pineal gland is more dense and fibrous than that of young animals. The distribution pattern ofthe pineal stroma shows variations in the course of adult life. This evolution, already indicated in pre­vious studies [Boya and Calvo, 1 977a, b; Calvo and Boya, 1977a-d], consists of a progressive fragmentation of the pineal parenchyma due to the penetration of thin connective sheets. In the pineals of the oldest animals studied (5

106 Boya/Calvo

Fig.l. 4-year-old hen o Compact aspect of the pineal parenchyma formed by many small pinealocyte

years old), the parenchyma appears divided in

small territories by thin connective septa

(fig.3). Most of these territories contain only one cellular rosette. The cellular component of these vascular-connective septa presents no variations with respect to the descriptions of of previous phases. The presence of lymphoid tissue in the pineal stroma of the adult phase is

worth to be pointed out.

Electron microscopy The pineal parenchyma of the adult hen

presents a solid aspect, using the low magnifi­cation of the eJectron microscope, due to the

rosettes. There are also a few larger formations of a more irregular arrangement (arrow). HE.

absence of follicular cavities typical of young animals [Boya and Calvo, 1977a, b). This

aspect remains al! along the age intervals studied. A more detailed study shows, how­ever, the existence of a great amount oflumina bordered by a line of junctional mechanisms

Fig.2. 3-year-old hen o Reaction to acid phosphat­ase. Note the abundance of Iysosomes and their ar­rangement in rosettes. The larger cavities are more scarce.

Fig . 3. 5-year-old heno Intense fragmentation of the pineal pareochyma in small territories limited by rhin connective-vascular septa. Gomori.

107 The pineal glaod in the adult chicken

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108 Boya/Calvo

Fig.4. 3-year-old heno WalJ of a smaJl cavity (L) systems, ciliary processes and terminal clubs. It is formed by a single layer of A (A) and B (B) pinealo­ limited by a series of junctional mechanisms (arrows). cytes. The cavity (L) is occupied by whorJ-like lamellar C = Connective-vascular septa. x 3,000.

The pineal gland in the adult chicken ]09

Fig.5. 5-year-old heno B pinealocyte. Golgi <':001­ Dense bodies (e). In the largest one, droplets or a O1ex in the vicinity of the nucleus (N). Presence of lipid aspect can be observed. x 15,000. plicrovesicles and larger dense-cored vesicles (arrows).

110 Boya/Ca lvo

(fig.4). Although these lumioa are totally occupied by the apical processes ofthe pinealo­

cytes, they are easily identified due to the

existence of a band of junctional mechanisms

that border the lumen . This explains the solid aspect observed at low magnifications. The pinealocytes are placed in a radial arrange­ment with respect to the lumina. The pattern

of radial arrangement is more regular as the diameter of the lumen beco mes larger. There

are also variations in the content ofthe lumina

in relation to their size. The lumina of larger diameter usually present whorl-like lame llar structures which are sometimes ver y well developed (fig.4, 10).

In the pinea! of the adu!t hen there is, be­tween the fol!icular and parafol!icular layers,

no parenchymal division as it 1S present in

young animals (fig.4) [Boya and Calvo, 1977a, b]. The parafollicular layer has disappeared. Therefore, due to the great number of lumina and the polarized aspect of the pinealocytes, we may admit that al! the parenchymal cells

are in contact with a central lumen (fig.4).

The stroma of the pineal gland of the adult hen is very rich in coJ]agen microfibri!s. Ex­cepting this Jarger fibrillar density, the con­nective tissue of the adult pinea! presents the same components (blood vessels, connective cel!s, unmyelinated nerve fibres, and Jymphoid

cells) as described in previous phases [Boya and

Calvo, 1 977a, b; CalvoandBoya, 1977a-d]. In addition to the thick connective septa, there are many, sometimes very thin, sheets of stroma distributed al! around the gland. These sheets tend to divide the pineal parenchyma in

very small territories, which contain a small

amount of lumina with their corresponding pinealocytes arranged radially. We frequently find pinealocytes whose nuelei (somas) are located in the vicinity of the basement mem­brane (fig.4). In any case, the distance between

the pinealocyte soma and the connective

space is always very smal! in adult p1neals. The pineal parenchyma of the adult hen

presents both cellular types, A and B pinealo­

cytes, which have been previously described in embryos and post-hatching stages [Boya and Calvo, 1977a, b; CalvoandBoya, 1977a-d].

The B pinealocyte is the most abundant cellular type (fig.4). These cells present a

marked hypertrophy which gives them a

globular aspect characteristic of this adult age. The nueleus is large and has a round or ovoid shape. It presents dispersed chromatin and several very prominent nuc!eoli (fig.4).

The cytoplasm of the B pinealocyte is very rich in organelles. The rough endoplasmic

reticulum 1S located in the vicinity of the nu­

cleus and in the peri phery of the a pical cyto­plasmo It appears in the form of long cisterns that place themselves in a pa rallel fashion. AlI the cytoplasm is very rich in free polyribo­somes (fig.4).

The Golgi complex is ver y well developed.

It is Jocated immediateJy aboye the nucleus.

Related to the Golgi complex we found numer­

Fig.6. A 5-year-old heno Large polymorphous dense bodies in a B pinea loeyte. Note the existenee of several lipid-Iike droplets in the Jargest one. )< 15,000. B 5-year-old heno Polymorphous dense bodies in the eytoplasm of a B pinealoeyte. The largest one presents a single, large lipid-like droplet. )< 20,000. e 4-year-old heno B pinealoeyte. LameJlar lipid fOlmed by a lipid­like central material (L) and a peripheral lamellar syslem (a rrows) wh ieh eorresponds to eisterns wi lhou t ribosomes. The spaee between this lamina is oeeupied by eytoplasmie bands. Note the development of lhe rough endopJasmie retieulum (RER) in the vieinity of the lamellar lipid and ils relation to it. )< 30,000. D 5-yea r-old heno B pinealoeyte. Lamellar lipid better limited than that in the 4-year-old hen (e) . Note the unpreeise limit of lhe lipid centre (L) and lhe periphe­ral membrane system free of ribosomes. x 20,000.

The pineal gland in the adult chicken J J 1

112 Boya/Calvo

ous clear vesicles as well as coated vesicles. There are also dense-cored vesicles of a larger diameter (800-1,500 Á). We have also found formations of these vesicles in the Golgi com­plex (fig.5).

The rest of the apical cytoplasm contains numerous mitochondria, which are more abundant than in previous phases (fig.4) . There are also microtubules in the entire cyto­plasm of the B pinealocyte.

The 'polymorphous dense bodies' are the most characteristic components in the B pinealocytes of the hen pineal (fig.4, 5, 10). They are located immediately aboye the Golgi complex interspersed among the mitochondria which are also located here. The polymorphous dense bodies ofthe adult pineals present round shapes . Only in those ofsmaller size do we find the irregular morphology which gives the name to those structures. They are considerably larger than those found in young animals . Most ofthem present a di ame ter near 1 ,um, although it is not rare to find larger dense bodies whose diameter lies between l.5 and 2 ,um. Their content is homogeneous and very electron­dense. During adult life, however, many poly­morphous dense bodies present several round droplets in their interior. These well-limited droplets are homogeneous and their content is less electron-dense than the rest of the body content (fig.5, 6a). These droplets are more frequent in the larger-sized bodies ; their size is approximately 0.2 ,um. Their shape and elec­tron density remind us ver y much of lipid droplets . Sometimes, these droplets seem to fuse in order to form one single droplet which is surrounded by all the rest of the electron­dense material (fig.6a). We have never found isolated homogeneous lipid droplets in the cytop1asm of the B pinealocytes. These dropJets have always been found in relation to the polymorpbous dense bodies.

The ultrastructural study of sections in­cubated for acid phospbatase proves the Iyso­somal nature of the polymorphous dense bodies, as has been described in previous papers [Calvo and Boya, 1977a-d]. In the in­terior of these dense bodies we found alead phospllate precipita te, perfectly visible even in contrasted sections (with uranyl acetate and lead citrate), which indicates the presence of acid phosphatase in them (fig.7, 8). But the aspect of this precipitate varies greatly among them. Mostly it appears in a homogeneous fashion occupying the whole interior of the particle. Other times we found thick granules scattered irregularly in the interior ofthe dense body (fig. 7). Only in some cases, we found one or two granules. Occasionally, we have found dense bodies with no enzymatic reaction . When the dense bodies present lipid droplets, they lack lead phosphate precipitate.

The Golgi complex of some B pinealocytes presents reaction product for acid phosphat­ase. However, in most of the B pinealocytes the Golgi complex lacks acid phosphatase activity. No other component of the B pineal­ocyte, excluding the aboye mentioned, pre­sents this type of activity.

Finally, in the cytoplasm of the B pinealo­cytes in the hen, we frequently find what we llave named 'lamellar lipids' (fig.6, 8) [Boya andZamorano, 1975 ; BoyaandCalvo, 1977a b ;

Fig.7. 3-year-old heno B pinealocyte. Polyrnor­phous dense bodies with a positive reaction to acid phosphatase. The lead phosphate precipitate appears in a hornogeneous fashion in sorne dense bodies. In others it appears in the fo rrn of granules distributed irregularly in the interior of the dense body (arro", x 30,000.

Fig. 8. 4-year-old hen o B pinealocyte. Pol. lIliY · phous dense body with a positive reaction to a . _ pbosphatase in reJation lo a lamellar lipid. x 20,000

113 The pineal gland in the adult chicken

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114 Boya/Calvo

Fig. 9. 4-year-old heno Basal process of a B pineal­ rows) and the existence of dense bodies (one of them ocyte (B) which ends in the vicinity of the basement is very large) and bars near the end of the process. membrane (MB) ¡hat limits the vascular-connective x 7,000. Inset: x 30,000. space (C). Note the abundance of microtubules (ar­

115 The pineaJ gland in the adult chicken

Fig.l0. 5-year-old heno SOlal1 cavily occupied which widens lo border Ihe cavily. It is relaled lo the largely by whorl-/ike lamellar systeOls (SL). In its wall adjacenl cytoplasOls by junctional OlechanisOls (ar­we Olay observe B pinealocytes (B) and Ihe supra­ rows). x 7,000. nuclear portion of a dense cell type A pinealocyle

116 Boya/Calvo

Calvo and Boya, 1977a-d]. They are Jocated in the immediate vicinity of the nucleus and, al­though they are generally single, there may be more than one per cel!. The size is difficult to determine since they lack clean limits, but we may say that the diameter ranges between 1.5 and 2,um.

The lamellar lipids are constituted by two components. The centre is occupied by a homogeneous material of irregular limits whose electron density reminds us of the lipid droplets described in the polymorphous dense bodies. Around this central component we find several concentric layers of a Jamellar aspect. In most of the lamellar lipids we find apparently empty spaces, enlarged and having unclear Jimits, which are found at the level of the concentric layers. These spaces seem to be responsible for the irregular contour of the central component. In the peri phery of the lame llar lipids, mainly the spaces between the lamina are occupied by cytoplasm in which we can sometimes distinguish cellular organelles (fig.6). On numerous occasions we have been able to demonstrate 1hat the peripherallamel­lar component corresponds to cisterns de­prived of ribosomes whose membranes have fused and the intracisternal space disappeared (fig. 6) . These cisterns proceed from the rough endoplasmic reticulum, although, on rare occasions, we have found images which sug­gest the origin of the cisterns in the Golgi complexo These last images are mainly found in the lamellar lipids of smaller size, apparently in the process of formation.

The organelle, most frequently related to the lamellar lipids, is the rough endoplasmic reticulum . These organelles place themselves in the form of parallel cisterns, but they never surround the 1amel1ar lipid nor do they place themselves concentrically to it. In the cisterns nearmost the lamellar lipids, we find images of

membrane fusion like those described pre­viously .

Less frequently, we found polymorphous dense bodies in contact with the lamellar lipids. However, we have not found images of fusion between both of these structures (fig. 8).

The B pinealocyte is a clearly polarized cell. This makes it possible to distinguish several regions in it, which we havepreviousJy de­scribed [Boya and Zamorano, 1975 ; Boya and Calvo, 1977a, b ; Calvo and Boya, 1977a- d] . The soma and the su pranuclear cytoplasm are very well developed, and therefore these cells have a typical globular aspect in the adult pinea!. The neck contains, as in previous phases, numerous microtubules which - pro­ceeding from the supranuclear cytoplasm ­assemble here. The junctional mechanisms are 10cated at this level (fig.4, 10). The terminal club presents a aspect similar to that found in young animal s, standing out the development of the ciliary processes (fig.10) , which appear swoJlen, and the whorl-like 1amellar systems related to them. The ultrastructure, course and location of the basal process present no ob­vious differences in respect to younger animals (fig.9) .

The A pinealocytes are much more scarce than the B pinealocytes in the adult pinea\. We may still observe the two cJear and dense cell types of the A pinealocytes [Boya and Calvo, 1977a, b], although the differences in density between them have greatly diminished. Thus, we find that the dense A cell type is only

Fig.ll. 4-year-old heno Dense type A pinealocyte. Dense bodies in relation lo a lipid droplel in the vi­cinity of a Golgi complex. C = Cilium. x 15,000.

Fig.12. 3-year-old heno A pinealocyle. Positive reaction to acid phosphatase in the dense bodies (ly­sosomes). x 20,000.

117 The pineal gland in Ihe adull chicken

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118 Boya/Calvo

slightly more electron-dense than the B pineal­ocyte . The cJear cell type, more abundant than the dense cell type, still presents a cytoplasmic

matrix which is almost electron-transparent.

The rest of the ultrastructural characteristics is similar in both clear and dense A cell types.

The morphology and relations of the A pinealocytes show no important variations with respect to that described for younger

animals [Boya and Zamorano, 1975; Boya and Calvo , 1977a, b; Calvo and Boya, 1977a-d].

The A pinealocytes have a scarce cytoplasm in comparison with the B pinealocytes, although is is more developed than in previous phases. The ovoid nucleus has a smaller size than that

of type B. The chromatin appears more con­trasted due to the lesser density of the nucleo­

plasmo It may present one or two nucleoli.

The A pinealocytes have a great relation to the pineallumina. At this level , they present a cytoplasmic apical swelling which is very characteristic (fig . 10). The luminal surface of

this cell presents short and little electron-dense microvilli as well as a cilium with a central pair

of microtubules. The basal process heads to­wards the basal membrane (fig.4) , at which level it ends in an enlargement.

In the pineal of the adult chicken, the A pinealocytes show a cytoplasm that is broader and more rich in organelles than in previous

phases. Among the organelles, the develop­ment ofthe Golgi complex and the presence of

numerous round and small (0.2-0.4 ,um) dense bodies are striking. They are located adjacent to the Golgi complex and in the vicinity of the luminal surface of the pinealocytes. In many

cases the dense bodies appear fused to a single

lipid droplet (fig. 11). This droplet, generally single, is surrounded by a thin surface of dense material. The lipid component is always much greater in size than the peripheral dense material.

The acid phosphatase technique, studied with the electron microscope, proves the pres­

ence of this hydrolase in the dense bodies of

the A pinealocytes which , therefore, must be

considered as Iysosomes (fig.1 2). The rest of the organelles in the A pinealo­

cytes is similar with respect to development and distribution to that described in young animals [Boya and Zamorano, 1975; Boya and Calvo , 1977a, b] . We may only emphasize the

presence of'nebenkern' in all the age intervals

studied in adult life. In addition to the already formed 'nebenkern' , we have found in the adult pineal images ofthe process of formation of these structures similar to those found in

young chickens.

Discussion

As is demonstrated by our studies , the ad ult chicken pineal gland presents a characteristic

morphology different to that observed in younger animals. In previous studies [Boya and Zamorano, 1975; Boya and Calvo, 1977a, b; Calvo and Boya, 1977a-d] we described the structure and ultrastructure of the chicken pineal along the embryonic development and

until13 months after hatching. With the pres­sent study we complete the description of the

pineal histology of this species. Recently, Omura [1977] has studied the ultrastructure of the chicken pineaJ during its embryonic devel­opment and during post-hatching life until 2 months of age. However, his description is centred almost exclusiveJy on the presence of

the dense-cored vesicles in the B pinealocytes,

paying little attention to the rest of the histo­logical characteristics of the pineal gland.

The structural pattern of the adult pineal gland represents a more advanced stage in the

119 The pineal gland in the adult chicken

evolution which began in early periods of chicken Jife . The details of this evolution have been described previously [Boya and Zamo­rano, 1975; Boya and Calvo, 1977 a, b]. The evolution of the pineal structural pattern refiects mainly the transformation of the Jarge follicular cavities into cellular rosette forma­tions. In the centre of these formations, which appear compact under the light microscope, there is a cavity perfectly visible under the electro n microscope. The larger cavities, which may be seen with the Jight microscope, cor­respond to the remains of the old follicular cavities which are thus still present in the adult pineal. The pineal cavities observed with the electron microscope appear occupied by the apical processes of the B pinealocytes . In all the age intervals studied, we frequently found whorl-like lamellar systems in the pineal lumina.

The arrangement ofthe pineal parenchyma in cellular rosettes is confirmed by the histo­chemical techniq ues for acid phosphatase which shows the pineallysosomes arranged in circular formations of lysosomal 'rosettes'. FinalJy, the progressive penetration of stroma into the parenchyma fragments it into small territories composed of one or several cellular rosettes. As a final result of this evolution process in the adult pineal , every pinealocyte is in contact witb a lumen and their somas are very near the basement membrane.

The tendency of the pineal to i ncrease its number of cavities, so that each cell may be in contact with one of them, indicates that this organization pattern is important for pineal physiology and not merely a vestigial reme m­brance. This is al so suggested by the cJear polarizatíon ofthe pinealocytes with respect to a central cavity. Also, the progressive penetra­tion of the stroma would result in a greater contact between the parenchyma and the

blood vessels and nerve fibres present in the stroma.

As shown by our ultrastructural results, the pineal gland is an active, functional organ duríng the adult life of the chicken. In effect, the cellular hypertropby and the great deveJ­opment of the organelles in the pinealocytes, especially in type B, are the indication of a cellular activity. Omura [1977] al so describes abundant organelles in this cellular type in 9­month-old chickens. The development of the rough endoplasmic reticuJum, Golgi complex, as well as of clear and dense-cored vesicles related to the Golgi complex, suggests an in­crease in the secretory activity ofthe B pinealo­cytes. Collin el al. [1976] have suggested, in autoradiographical studies , the location of índoleamines in the dense-cored vesicles ofthe avían pinealocytes. The presence ofthe numer­ous mitochondria in the B pinealocyte would provide the necessary energy for an increased cellular activity .

From the morphologic point of view, the activity of the pineal gland must remain at a constant level during adult life. This may be demonstrated by the uniformity of the ultra­structural pattern of the pineal parenchymal cells in all the age intervals studied (1-5 years of age).

FinaJly, in none ofthe age intervals studied with the electron microscope we have found any images which indicate the appearance of degenerative or regressive phenomena in the pineal gland. The involution of the hen pineal gland must, therefore, take place in a stage aboye 5 years.

One the most characteristic organelles of the B pinealocytes is the polymorphous dense body [Boya and Zamorano, 1975 ; Boya and Calvo, 1 977a, b; Calvo and Boya, 1977a-d]. In the adult pineal, these structures are much larger and more abundant than in the pineal

120 Boya/Calvo

of young animals. Thus, this hypertrophy of the polymorphous dense bodies may be con­sidered as a specific ultrastructural charac­teristic of the adult pinea!. With the data available to us we cannot explain the signifi­cance ofthe hypertrophy ofthe polymorphous dense bodies .

In a previous paper [Calvo and Boya, 1977a-d] we demonstrated the Iysosomal nature oftbe dense bodies in the B pinealocytes due to the presence of acid phosphatase in them. The hypertrophic dense bodies of the adult pinealocytes also present acid phosphat­ase activity, although the precipitate is not located in a uniform fashion in all the dense bodies. The presence of a reaction product for acid phosphatase located in an irregular fashion is usually found in secondary Iyso­somes. Morphologically, the polymorphous dense bodies of the adult pinealocytes do not present the typical heterogeneous aspect of these secondary Iysosomes, although their hypertrophy could be a positive support for this idea.

The lamellar lipids are characteristic struc­tures of the B pinealocytes. According to our results, they are present from the embryonic period until adult life. Omura [1977] describes a juxtanuclear lipld inclusion in pinealocytes of 21-day embryos. Its aspect corresponds to what we have named lamellar lipids. The detailed study ofthese structures demonstrates the formation origin ofthe peripherallamellar component to be mainly the rough endo­plasmic reticulum. A review of the material corresponding to younger animals confirms this idea.

We have found numerous images in the adult B pinealocytes which suggest the origi n of the lamellar lipids to be the polymorphous dense bodies. The evolution process could follow a series of steps: (a) appearance of

homogeneous droplets of small electron den­sity, having a probable lipidic nature in the polymorphous dense body; (b) fusion of these dropJets to constitute a single large one which occupies a great part of the dense body volume, and (c) placement of strands proceed­ing from the fusion of the membranes of the rough reticulum cisterns around the lipids and , thus, appearance of the lamellar lipid. We must, however, make clear the following points: (1) this explanation is based only on morphological observations , having no defi­nite proofs of its validity in our material. (2) Certain aspects are not explained, such as the destiny of the dense material of the dense body or the negativity of the lame llar lipids to the techniques for acid phosphatase. We would also have to make clear why, although the number of polymorphous dense bodies is very abundant, we only found one or two lamellar lipids per cell. (3) This diagram can only be applied to adult pineals since the polymor­phous dense bodies of pineals of young ani­mal s do not present lipid droplets. Further investigations are necessary in order to proof this hypothesis and to obtain data about the formation of lamellar lipids in embryos and young animals.

The A pinealocytes during adult life a re large cells rich in organelles with respect to those observed in younger animals. This cellula r type has been regarded as a supporting cell by several authors [Oksche and Vaupel van Harnack, 1966 ; Fujie, 1968 ; Ca lIin , 1971 ; Omura, 1977] and also as an ependymal ce.ll [Bischojf, 1969]. The function of mechanical support, ifsuch, must not be ver y important if we are to judge by the amount of microtubules and microfilaments present in this cellular type. Instead, its relation to ependymal cells is more evident. In effect. the study of the choroid epithelium reveals tbe presence in its

121 The pinea! gland in the adu!t chicken

cells of round dense bodies, oftea related to a lipid droplet, similar to those present in A pinealocytes. Also, the microvilli, typical of the A pinealocyte, are similar to those of the choroid epithelium. Finally, the cells of the choroid plexus present cilia as in the A pinealo­cytes.

In previous papers [Boya and Zamorano, 1975 ; Boya and Calvo, 1977a, b; Calvo and Boya, 1977a-<i] we have described the relation between the A pinealocytes and the follicular lumina in formation. In the adult pineal, the developmenr of organelles in these cells could indicate a metabolic or nutritional function of the A pinealocyte.

References

Bischoll M.B.: Photoreceptora! and secretory struc­tures in the avian pinea! organ. J. Ultrastruct. Res. 28: 16- 26 (1969).

Boya, J. anJ Calvo, J.: Post-hatching evolution of the pineal gland of the chicken. Acta anal. (in press, 1977a).

Boya, J. and Calvo, J.: Ultrastructural study of the post-hatching evolution of the pineal gland of the chicken ( Gal/lIs gal/lIs). Acta anat. (in press, I 977b).

Boya, J. and Zamorano, L.: Ultrastructural study of the pineal gland of the chicken (Galllls gol/lis). Acta anal. 92: 202-226 (975).

Calvo, J. and Boya, J.: Embryonic development of the pinenl gland of lhe chicken. Acta anat. (in press, 1977a).

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122 Boya/Calvo

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Received: January 11, ]978

J .Boya, Department ofHistology and Embryology, Faculty of Medicine, University Complutense, Madrid (Spain)